📄 agriculture

Quantitative modelling of survivability and controlled release of a double-entrapped Azospirillum formulation

This study demonstrates that a double-entrapped Azospirillum formulation, combining alginate beads within a non-woven biodegradable matrix, significantly enhances bacterial storage survivability, enables controlled release, and improves plant growth and yield under both normal and salt-stress conditions compared to conventional carrier systems.

Original authors: Abitha Benson, Melvin Joe Manoharan, T Ragunathan, R. Nagajothi, M Selvarani, Denver I. Walitang, Tongmin Sa

Published 2026-08-19

📖 5 min read🧠 Deep dive

CC BY 4.0

Original authors: Abitha Benson, Melvin Joe Manoharan, T Ragunathan, R. Nagajothi, M Selvarani, Denver I. Walitang, Tongmin Sa

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

In the vast fields of agriculture, farmers have long relied on chemical fertilizers to feed crops, but these synthetic inputs come with a heavy environmental price tag, contributing to pollution and climate change. Nature offers a cleaner alternative in the form of tiny, beneficial bacteria that live in the soil and help plants grow by unlocking nutrients and producing natural growth hormones. One such bacterium, called Azospirillum, is particularly valued for its ability to help plants like rice thrive without heavy chemical assistance. However, for decades, these living helpers have been difficult to use effectively. When farmers try to apply them, the bacteria often die quickly because they are too fragile to survive the harsh conditions of storage or the sudden shock of being dropped into dry soil. They need a safe place to live and a way to be released slowly, but finding a container that protects them without trapping them inside has been a persistent challenge for scientists.

A team of researchers set out to solve this problem by creating a new kind of protective home for these bacteria, one that combines two different materials to create a double layer of defense. Instead of simply mixing the bacteria into a gel or wrapping them in a single sheet, they developed a system where tiny beads of a natural, jelly-like substance called alginate, which hold the bacteria, are carefully tucked inside a piece of non-woven paper. This paper acts like a sturdy, breathable jacket that shields the delicate beads from physical damage and harsh environmental shifts. The researchers wanted to see if this double-entrapped design could keep the bacteria alive longer during storage and release them more steadily into the soil compared to the traditional single-layer methods currently in use. They tested their creation by watching how the bacteria survived over time, measuring how fast they escaped the carrier, and finally planting them in soil to see how well they helped rice plants grow, even when the soil was salty and stressful.

The results showed that this double-layer approach worked significantly better than the older methods. When the researchers tracked the bacteria over a period of six months, they found that the double-entrapped system kept the bacteria alive much longer. In the traditional carriers, the bacteria died off quickly, but in the new double-layer design, the population declined very slowly, with the rate of death dropping to just 0.003 per day in the final stage of storage. This suggests that the outer paper layer provided a crucial buffer, shielding the bacteria from the stress of drying out or temperature changes. The study also looked at how the bacteria moved out of the carrier and into the soil. Using a mathematical approach to track the release, they found that the double-entrapped system released the bacteria at a steady, controlled pace, rather than all at once. This slow release is exactly what is needed for the bacteria to establish themselves in the soil and help the plant roots.

When the researchers moved from the lab to actual pots of soil, the benefits of this new design became even clearer. They grew rice plants using the double-entrapped bacteria and compared them to plants grown with the traditional carriers or no bacteria at all. The plants receiving the double-entrapped treatment grew taller, developed larger leaves, and produced more grain than the others. In normal soil, the rice plants grew to a height of 100 centimeters, which was 15 percent taller than those treated with the standard alginate beads and 33 percent taller than those with the non-woven paper alone. Even more impressively, when the researchers simulated salty, difficult soil conditions that usually stunt plant growth, the double-entrapped bacteria still helped the rice plants outperform the others. The plants in this group had roots that were 20 percent longer and produced 25 percent more grain than those treated with the traditional alginate method.

The study also measured the biological activity of the bacteria in the soil, finding that the double-entrapped system led to higher levels of nitrogen fixation, the process by which the bacteria convert air into a form of nitrogen that plants can eat. This activity was 35 percent higher than in the control group with no bacteria. The researchers observed that the bacteria were able to colonize the roots of the rice plants more effectively, sticking to the root surfaces in dense clusters rather than washing away or dying off. This strong presence in the root zone, known as the rhizosphere, is what drives the improved growth and yield. By creating a carrier that protects the bacteria during storage and releases them slowly into the soil, the researchers have demonstrated a way to make these natural plant helpers more reliable and effective. This approach could help farmers reduce their reliance on chemical fertilizers while still achieving high crop yields, even in challenging environments.

Technical Summary: Quantitative Modelling of Survivability and Controlled Release of a Double-Entrapped Azospirillum Formulation

Problem Statement
Bioinoculants, particularly Azospirillum-based biofertilizers, face significant limitations in global adoption due to poor survivability during storage and rapid population decline upon soil application. While alginate-based encapsulation is a common strategy for microbial protection, it suffers from low mechanical strength and restricted tolerance to environmental stresses, leading to uncontrolled diffusion and rapid release. Conversely, non-woven materials offer superior mechanical strength but lack systematic investigation for Azospirillum formulation. Furthermore, there is a lack of quantitative understanding regarding the survivability and release kinetics of these formulations using mathematical models, specifically biphasic survival models and diffusion-reaction analyses.

Methodology
The study developed a double-entrapped (DE) carrier system by embedding Azospirillum-containing alginate beads (approx. 3 mm diameter) within layers of non-woven biodegradable paper (abaca), creating a composite structure. The non-woven paper was treated with 5% glycerol to restrict oxygen diffusion, and the assembly was sealed with gum Arabic.

The research employed a multi-faceted approach:

Storage Survivability: Bacterial viability was monitored over 180 days using the Most Probable Number (MPN) technique on nitrogen-free malic acid medium.
Mathematical Modelling:
- Biphasic Survival Model: Used to fit storage data, distinguishing between fast ( $N_1, k_1$ ) and slow ( $N_2, k_2$ ) inactivating populations.
- Diffusion-Reaction Analysis: Effective moisture diffusivity ( $D_{eff}$ ) was calculated using Fick's second law. The Thiele modulus ( $\Phi$ ) and Biot number ($Bi$) were computed to characterize transport regimes (reaction-controlled vs. diffusion-controlled).
- Release Kinetics: The Weibull model was applied to analyze bacterial release rates under normal and salt-affected conditions (100 mM NaCl).
In Vivo Evaluation: Pot culture experiments were conducted using rice (Oryza sativa L. cv. Green World Pusa PR-130) under greenhouse conditions. Treatments included uninoculated controls, free PGPR, alginate-based formulations, and the DE formulation. Parameters measured included root architecture, nitrogenase activity, plant height, dry weight, leaf area, and grain yield.
Statistical Analysis: Data were analyzed using ANOVA, Duncan's Multiple Range Test (DMRT), and Principal Component Analysis (PCA) to evaluate relationships between carrier properties and biological performance.

Key Results

Survivability: The DE formulation demonstrated superior storage stability compared to alginate and non-woven paper alone. The biphasic model revealed the lowest inactivation rate constants for the DE system ( $k_1 = 0.030$ day $^{-1}$ and $k_2 = 0.003$ day $^{-1}$ ), representing a 57% reduction in fast inactivation and a 70% reduction in slow inactivation compared to the non-woven carrier.
Transport Dynamics: Moisture diffusion analysis indicated that the DE system operates under a diffusion-controlled transport regime. It exhibited the highest normalized effective diffusivity ( $D_{eff}$ ) with a relative value of 1.0 (compared to 0.14 for non-woven and 0.37 for alginate), an absolute $D_{eff}$ of $2.57 \times 10^{-9}$ m $^2$ day $^{-1}$ , a Thiele modulus of 6.84, and a Biot number of $6.73 \times 10^5$ . These values confirm that the composite matrix provides a strong diffusion barrier, protecting the cells at the cost of internal mass transfer limitations.
Release Kinetics: The Weibull model confirmed a sustained, controlled release profile for the DE system. Under normal soil conditions, the release rate constant ( $k$ ) was $0.135$ day $^{-1}$ , which was 18% slower than alginate and 52% slower than non-woven paper. This trend persisted under salt stress, where the DE system maintained a lower release rate ( $k = 0.235$ day $^{-1}$ ) compared to other carriers.
Plant Growth and Colonization: In pot culture studies, the DE formulation resulted in significantly higher rhizosphere colonization compared to other formulations. Specifically, the DE carrier recorded a 26% higher population compared to alginate and a 50% higher population compared to non-woven carriers under normal conditions. Under normal conditions, DE-treated rice showed a 16% increase in root length and a 30% increase in root area compared to alginate. Under salt stress, DE treatment maintained higher root lengths (5% higher than alginate) and significantly improved grain yield (4.5 g plant $^{-1}$ ), which was 25% higher than alginate and 67% higher than the control. Nitrogenase activity was also highest in the DE treatment under both normal and salt-stressed conditions.

Significance and Claims
The paper claims that the double-entrapped carrier system successfully addresses the trade-off between mechanical protection and controlled release in biofertilizer formulations. By combining alginate beads with a non-woven matrix, the system creates a protective microenvironment that significantly extends bacterial shelf life and modulates release kinetics. The study asserts that this diffusion-controlled protection enhances microbial persistence in the rhizosphere, leading to improved root architecture and plant growth, particularly under abiotic stress conditions like salinity. The integration of quantitative modelling (biphasic survival, Thiele modulus, and Weibull kinetics) provides a robust framework for understanding and optimizing the transport phenomena within PGPR carriers, moving beyond empirical observation to a mechanistic understanding of formulation performance.

More like this